Comprehensive functional analysis of chymotrypsin C (CTRC) variants reveals distinct loss-of-function mechanisms associated with pancreatitis risk

Abstract

Objective The digestive enzyme chymotrypsin C (CTRC) protects against pancreatitis by promoting degradation of trypsinogen and thereby curtailing potentially harmful trypsinogen activation. Loss-of-function variants in CTRC increase the risk for chronic pancreatitis. The aim of the present study was to perform comprehensive functional analysis of all missense CTRC variants identified to date. Design We investigated secretion, activity and degradation of 27 published and 5 novel CTRC mutants. We also assessed the effect of 5 mutants on endoplasmic reticulum (ER) stress. Results None of the mutants exhibited a gain of function such as increased secretion or activity. In contrast, 11 mutants showed marked loss of function, 3 mutants had moderate functional defects, whereas 18 mutants were functionally similar to wild-type CTRC. The functional deficiencies observed were diminished secretion, impaired catalytic activity and degradation by trypsin. Mutants with a secretion defect caused ER stress that was proportional to the loss in secretion. ER stress was not associated with loss-of-function phenotypes related to catalytic defect or proteolytic instability. Conclusion Pathogenic CTRC variants cause loss of function by three distinct but mutually non-exclusive mechanisms that affect secretion, activity and proteolytic stability. ER stress may be induced by a subset of CTRC mutants but does not represent a common pathological mechanism of CTRC variants. This phenotypic dataset should aid in the classification of the clinical relevance of CTRC variants identified in patients with chronic pancreatitis.

Pathogenic cellular role of the p.L104P human cationic trypsinogen variant in chronic pancreatitis

“…Thus, high-penetrance PRSS1 variants cause higher trypsin levels either by stimulating trypsinogen autoactivation and/or by inhibiting CTRC-dependent trypsinogen degradation (13,33). Loss-of-function mutations in the protective trypsin inhibitor SPINK1 or the trypsinogen-degrading enzyme CTRC can also result in elevated trypsin activity (1,4,5,15,18,26). Consistent with the trypsin paradigm, a rapidly autodegrading variant of PRSS2 encoding human anionic trypsinogen was shown to protect against chronic pancreatitis (47).…”

Genetics of Pancreatitis: The 2014 Update

“…CPA1 had no detectable effect on trypsinogen activation, trypsin activity or trypsinogen degradation by CTRC, indicating that CPA1 variants do not exert their effect via increasing the intrapancreatic trypsin activity. On the other hand, most of the loss-of-function CPA1 variants exhibit markedly reduced secretion, raising the possibility that CPA1 variants undergo misfolding in the ER and cause ER stress, as demonstrated previously for some PRSS1 and CTRC mutants (Kereszturi et al 2009b;Beer et al 2013). Indeed, the expression of p.N256K variant in AR42J rat pancreatic acinar cells resulted in ER stress, as evidenced by increased splicing of X-box binding protein 1 and elevated mRNA levels of the chaperons BiP and calreticulin.…”

“…It has been reported that the CTRC variants in exons 3 and 7 are associated with CP in Europe and India (Rosendahl et al 2008;Masson et al 2008;Beer et al 2013;Paliwal et al 2013). In European cohorts, the micro-deletion variants p.K247_R254del (c.738_761del24) and p.R254W, both located in exon 7, are the most common CTRC variants (Rosendahl et al 2008;Masson et al 2008;Beer et al 2013). In India, these variants are rare, and the p.A73T (c.217G>A) variant in exon 3 and the p.V251I (c.703G>A) variant in exon 7 are the most common ones (Rosendahl et al 2008;Paliwal et al 2013).…”

Hereditary Pancreatitis

“…Loss-offunction mutations in chymotrypsinogen C (CTRC) and the serine protease inhibitor, Kazaltype 1 (SPINK1) reduce the protective functions of these trypsin inhibitors in the pancreas (7,52,68,84).…”